Standardization of Synthetic Biology Tools and Assembly Methods for Saccharomyces cerevisiae and Emerging Yeast Species

Abstract

As redesigning organisms using engineering principles is one of the purposes of synthetic biology (SynBio), the standardization of experimental methods and DNA parts is becoming increasingly a necessity. The synthetic biology community focusing on the engineering of Saccharomyces cerevisiae has been in the foreground in this area, conceiving several well-characterized SynBio toolkits widely adopted by the community. In this review, the molecular methods and toolkits developed for S. cerevisiae are discussed in terms of their contributions to the required standardization efforts. In addition, the toolkits designed for emerging nonconventional yeast species including Yarrowia lipolytica, Komagataella phaffii, and Kluyveromyces marxianus are also reviewed. Without a doubt, the characterized DNA parts combined with the standardized assembly strategies highlighted in these toolkits have greatly contributed to the rapid development of many metabolic engineering and diagnostics applications among others. Despite the growing capacity in deploying synthetic biology for common yeast genome engineering works, the yeast community has a long journey to go to exploit it in more sophisticated and delicate applications like bioautomation.

Keywords: automation; biological parts; characterization; standardization; synthetic biology; yeast toolkits.

Figure 1

Overview of the selected yeast synthetic biology toolkits mapped in this review.

Figure 2

Standard biological parts listed for S. cerevisiae at the iGEM Registry.

Figure 3

Hierarchical assembly strategy in MoClo YTK. (A) Golden Gate-based assembly mechanism of the toolkit. The parts are first generated via PCR or synthetic DNAs are used as sources and they are kept in the part plasmids. In the next level, the parts are assembled by using the BsaI type IIS restriction enzyme to create transcriptional units (TU) that usually contain a promoter, coding sequence (CDS), and terminator. At this level, plasmids have an ampicillin-resistance marker (AmpR). When needed, multiple TUs can be assembled by using the BsmBI type IIS restriction enzyme to obtain a multigene plasmid. At this level, plasmids have a kanamycin-resistance marker (KanR). (B) The part types used in MoClo YTK. Each number represents a particular type. The types can be further modularized. Type 3 can be split into two so that an N-terminal tag (Type 3a) can be used with the CDS (Type 3b). Likewise, Type 4 can be either a C-terminal tag (Type 4a) or terminator (Type 4b) for genomic integration, and Type 7, which is used for yeast origin of replication (ORI), can be replaced with a 3′ homology arm, where Type 8b can be used as a 5′ homology arm. Then, the construct is linearized with homology arms at each end.

Figure 4

The overall scheme of multi-gene constructs via the YeastFab method. The functional YeastFab parts can be cloned into part accepting vectors by using the BsaI type IIS restriction enzyme. These domesticated parts can be released from part accepting vectors by using Esp3I (BsmBI) type IIS restriction enzyme and transcription units are assembled in a POT accepting vector in a promoter-ORF-terminator grammar. Following this, several transcription units in multiple POT vectors can be assembled together using BsaI.

Figure 5

Representation of the GoldenBraid (GB) assembly method. (A) The modules, promoter (PRO), coding sequence (CDS), terminator (TER) are assembled into the level Ω (with spectinomycin-resistance gene SpR) or level α entry vectors (with kanamycin-resistance gene KanR) depending on the restriction enzyme used. The PC fragment between the PRO and CDS connects these two parts after being cut by a proper type II restriction enzyme. Similarly, the CT fragment connects the CDS and TER. Then, the transcription unit (TU) consisting of these three parts is assembled into an entry vector. LACZ is used as a reporter to detect the correct assemblies. (B) Two TUs can be assembled from level Ω to level α vectors or vice versa. Depending on the type II restriction enzyme used, the next level is selected. 1, 2, 3 or A, B, C in ellipse shapes represent inner cutting sites of the type II restriction enzymes so that a common sticky end can be formed with the next TU. In the first assembly, two single TUs share a common “C” sticky end, whereas “3” is shared by two double TUs in the next assembly step. More TUs can be assembled by following this order or reused as entry vectors for the next level α binary assembly. They both have a BsaI sticky end. (C) Yeast GB assembly approach used for yeast. TU contains an additional N-terminal tag for mitochondrial targeting. Also, 5′ and 3′ homology arms are added for genomic integration into the target region. The construct is linearized by the I-SceI type II restriction enzyme.

Figure 6

Working principle of Cas9-based pCut toolkit assembly method. (A) CASdesigner software can be used to design DNA oligos for the target construct. Donor DNA can be assembled to form a complete donor or the parts (promoter, PRO; coding sequence, CDS; terminator, TER) containing short homology fragments to adjacent fragments can be cotransformed for in vivo assembly. C-terminal localization signals (TAG) can be also used in donor DNA construct. (B) Cas9 and gRNA are expressed by CRISPR plasmid, and they lead to double-strand break formation on the target region of the yeast genome. Following that, donor DNA is integrated via homology-directed repair thanks to 5′ and 3′ homology arms.

Figure 7

Toolkits adapted from MoClo YTK for K. phaffii and K. marxianus. Additional parts (shown on green background) including secretion signals (TAG), promoters (PRO), and terminators (TER) have been characterized for K. phaffii and the parts are provided with the MoClo Pichia toolkit. Additionally, K. marxianus-specific parts (shown on blue background) containing 19 promoters and 5 terminators have been characterized. Alternatives of yeast origin of replications (ORI) have also been added to the kit (K. marxianus kit, KmK) along with four homology arms for genomic integration of the constructs (HA, homology arm; Chr, chromosome; MF, mating factor).

Figure 8

GoldenMOCS assembly system and the relevant toolkits. (A) Similar to MoClo, the basic modules (promoter, PRO; coding sequence, CDS; terminator, TER) are kept in BB1 vectors and they are assembled using BpiI type II restriction enzyme to create complete transcription units (TU) in BB2 vectors. Multigene constructs can be created via BsaI type II restriction enzyme into BB3 vectors. (B) Species-specific parts have been characterized for K. phaffii (GoldenPics) and Y. lipolytica (GoldenMOCS-Yali) using the GoldenMOCS assembly approach.

Figure 9

Sample overhang misannealing analysis of Tatapov used for yeast GB toolkit. (A) General evaluation of overhang misannealings in the target set. The squares outside the diagonal square pairs show the cross-talking, so misannealing risks. In addition, lighter square pairs on the diagonal show weak self-annealings, so the risk of having no assembly. (B) Detailed evaluation for the CTCC overhang.